The complement (C) system is an important element in the innate immune system and functions as an enzyme cascade system and involves a large number of distinct plasma proteins and membrane proteins that react with one another in sequence to opsonize invading micro-organisms, to directly kill micro-organisms, and to induce an immediate series of inflammatory responses that help fight infections. The innate immune system differs from the part of the immune system termed the adaptive immune system, in that it does not confer long-lasting or protective immunity to the host, it is however important to stress that there are important links between the two parts of the immune system. The adaptive immune system is further divided into humoral and cellular components.
Uncontrolled activation of the complement system would result in damage to host cells. To prevent that the complement system for example is activated on host cells the system is regulated by regulatory complement factors either soluble plasma proteins or membrane bound proteins.
There are three distinct pathways through which the complement system can be activated. These pathways are termed the classical complement pathway (CP), the alternative complement pathway (AP), and the lectin complement pathway (or the mannan-binding pathway) (LP), and depend on different molecules and mechanisms for their initiation, but they converge to activate the same central effector molecule, C3, leading to fragments C3b and C3a.
The AP is initiated when C3b binds to a non-self surface, whereby cleavage and inactivation by the regulatory complement factor I is avoided. The AP initiation involves the complement factors C3b, factor B, factor D and Properdin.
The CP is initiated when multivalent binding of C1q to antigen-antibody complexes (immunocomplexes) is achieved, and involves the factors C1r, C1s, C2 and C4.
The LP is an ancient constituent of the complement system, probably predating the CP, and shares C2 and C4, with CP. However, initiation of the lectin pathway is antibody-independent with the difference lying in the initiation complex. The lectin pathway is activated when a collectin (MBL or ficolin) recognizes conserved patterns of carbohydrate structures on the surface of various microorganisms and furthermore involves MBL-associated serine proteases (MASP's).
MBL is an acute-phase protein; a modest 1.5 to 3 fold increasing in serum MBL concentration is seen after malaria infection and trauma (e.g, surgery). MBL is an oligomeric protein with an overall bouquet-like structure, resembling in this aspect C1q, and it belongs to the collectin family of proteins that consist of a collagen-like domain, an alfa coiled coil helical neck region and a carbohydrate recognition domain (CRD). It is through the CRD domain MBL exerts its functions by binding to carbohydrate structures on bacterial or viral surfaces.
Binding of MBL to a surface may by itself result in e.g. a microorganism being opsonized (facilitate fagocytosis), however, the activation of the lectin complement pathway is through activation of serine proteases (MASP's) linked to MBL (or ficolin). Four such serine proteases have been described, MASP1, MASP2, MASP3, and sMAP (sMAP is lacking a protease domain). In a recent study it has been demonstrated that in serum, proenzyme MASP-2 is entirely associated with MBL, whereas MASP-1 circulates in both bound and in unbound forms. The overall structures of MASPs resemble C1r and C1s and mimic their activities. Upon binding of MBL to carbohydrate structures on the surface of microbes, the proenzymes of MASP's are cleaved resulting in the active form. The activated MASP's are then able to exert their proteolytic activities against complement components. MASP-2 activates C4 and C2, and form the C3 convertase (C4bC2a) an effect similar to the effect of the CP C1s. Although MASP-1 is a more efficient peptidase than MASP-2, it does not display any efficient complement activation cleavage. However, it has been suggested that MASP-1 is involved in the complement activation through the direct cleavage of C3, but independent in vitro experiments showed that this is very unlikely. To date the exact function of MASP-3 and sMAP are unknown. It has been proposed that they may function as regulators of the MBL pathway activation through competitive inhibition of MASP-2 binding to MBL.
Serum concentrations of MBL are extremely variable ranging from almost 0 to 5 μg/ml in healthy humans. The low MBL concentrations are a consequence of polymorphism in the promoter region and/or in the structural part of the MBL gene.
The complement system is a major part of the immune defense against invading microorganisms, in particular during the early phase after pathogen entry. The AP and the LP will immediately be activated when foreign surfaces are recognized within the host. Together with other elements of the innate immune system recognition of “foreign” is also an essential element in the subsequent transport of microbial antigens to secondary lymphoid organs where lymphocyte stimulation occurs, leading to activation of the adaptive immune response with production of antibodies and effector T-lymphocytes as effector systems. Taken together, the complement system is required for optimal function of the host immune system.
Defects in the complement system may for example be due to genetic defects arising from mutations in the genes coding for individual complement components, or may for example be acquired defects, e.g., caused by excessive consumption of complement due to the presence of immune complexes or activators of AP (e.g. endotoxin), or caused by the presence of autoantibodies to complement factors, e.g. nephritic factor. Such defects in the complement system may result either in complete blockade of the involved complement pathway or in partial blockade leading to impaired function. Defects in particular components may also in some cases lead to impaired effector function of two or all three pathways.
Most deficiencies in the complement system may lead to diseases ranging from increased susceptibility to infections, to severe autoimmune manifestations etc. It is therefore of considerable clinical importance to be able to monitor the functions of the complement system, both as a diagnostic tool, but also as a prognostic marker and as a marker of disease activity.
Undesired activation of the complement system may lead to inflammation and tissue damage and can for example be seen in conditions such as autoimmune diseases, immune complex diseases, conditions with ischemia and in the course of host versus graft or graft versus host reactions.
Functional deficiencies in the lectin pathway are usually due to genetic polymorphisms in the MBL gene, but recently, deficiencies in the MASP genes and polymorphisms of Ficolin genes have also been reported. In general, genetic deficiencies of the complement system components are rare; an exception to this is MBL deficiency, which in human populations may occur with frequencies of 5-7%.
MBL deficiency is the most common inherited immunodeficiency (MBL concentration <100 ng/ml) with a prevalence of 5-7% in the general population. MBL deficiency has been investigated in many different populations and is largely explained by three single point mutations in codons 52, 54 and 57 of exon 1 of the MBL gene. These mutations are also frequently referred to as variants D, B and C, respectively, with variant A representing wild type. These mutation-frequencies are different among populations. In Eurasian populations the B variant mutation make up 22-28%, whereas the C variant mutation is characteristic of sub-Saharan African populations making up 50-60%. Finally, the D mutation makes up 14% in European populations. A common result of the mutations in the exon 1 MBL gene is an impaired oligomerization, which leads to a functional deficiency of the MBL protein. Several studies show that MBL deficiency is associated with increased susceptibility to many infectious diseases, namely extracellular pathogens and particularly organism which cause acute respiratory tract infections during early childhood. However, studies also indicate that pathology arising from MBL deficiency may require one or more co-existing immune deficits. For example, recent studies on Neisseria menigitidis disease show an increased probability of the disease when MBL deficiency is associated with a properdin defect. In order to give the correct and most efficient treatment in relation to disorders, conditions, diseases and/or genetic deficiencies in the immune system, it is of paramount importance to identify where the e.g. disorder is located.
In the past, complement function has been measured through haemolytic assays which enable functional assessment of the classical (CP) and the alternative (AP) complement pathways through their ability to generate the membrane attack complex (C5b-9) upon activation. Similar assays for the lectin pathway, are currently not available. In general, the available assays are cumbersome and tedious, and more easy-to-perform assays are required.
A similar assessment of the lectin complement pathway function is hampered by interference from activation of the classical and the alternative complement pathway. Activation of the lectin complement pathway requires an activating surface with carbohydrate structures, and most often there will in the activation phase be interference from anti-carbohydrate antibodies from the test sample giving rise to activation of the classical complement pathway, and from a direct activation of the alternative complement pathway due to the presence of a non-self surface. Therefore, a reliable assay must prevent the concomitant activation of these two pathways.
Until now, at least three assays have been described for determining functional deficiencies in the lectin complement pathway. One assay measures the functional activity of lectin-MASP complex through activation of added exogenous C4. Another assay measures endogenous activation of C4 after blocking of classical and alternative complement activation by using a high ionic strength dilution buffer (1M NaCl) (Petersen S V, Thiel S, Jensen L, Steffensen R, Jensenius J C.: An assay for the mannan-binding lectin pathway of complement activation. J Immunol Methods. 2001; 257:107-16). Finally, a lectin pathway assay uses a monoclonal antibody to C1q to inhibit classical complement activation, and activation of the alternative complement pathway is inhibited either through dilution or by an antibody to complement factor D (Roos A, Bouwman L H, Munoz J, Zuiverloon T, Faber-Krol M C, Fallaux-van den Houten F C, Klar-Mohamad N, Hack C E, Tilanus M G, Daha M R.: Functional characterization of the lectin pathway of complement in human serum. Mol Immunol. 2003; 39:655-68).
In the previously known methods the complement pathways are for example severely influenced by the use of 1M NaCl buffers, and the use of a monoclonal antibody to C1q does not exclude interactions via the AP. Furthermore, the previously known methods are in general cumbersome, expensive and influenced by the AP and the CP. Accordingly, an easy-to-perform and reliable method for determining potential functional deficiencies in the lectin pathway of the complement system is needed.